USPTO Patents Application 10687850

Europaisches Patentamt
European Patent Office
Office europeen des brevets

© Publication number:

0 241 309

A2

©

EUROPEAN PATENT APPLICATION

© Application number: 87303166.0
@ Date of filing: 10.04.87

tnt.ci.<: G 01 N 33/53

G 01 N 27/00, C 12 Q 1/00,
C 12 M 1/40

© Priority: 10.04.86 GB 8608700

©

Applicant: Genetics International, Inc.

128 Sidney Street

@ Date of publication of application :

Cambridge Massachusetts 02139 (US)

14.10.87 Bulletin 87/42

@

Inventor: Walton, Nicholas John

@ Designated Contracting States:

22 Summerfleld

CH DE FR GB IT U

New Hlnksey Oxford OX1 4RH (GB)

Chambers, Gill Alison

159Flatford Place

KidHngton Oxford OX5 1TG (GB)

®

Representative: Ruffles, Graham Keith et al

MARKS & CLERK 57-60 Lincoln's Inn Fields

London WC2A3LS (GB)

CM
<

o

CO

The application Is published incomplete as filed (Article 93 (2) EPC) . The point in the description or the claim(s) at which the
omission obviously occurs has been left blank. Page 15, line 16

A request for correction of page 15 of the description has been filed pursuant to Rule 88 EPC. A decision on the request will be
taken dunng the proceedings before the Examining Division (Guidelines for Examination in the EPO, A-V, 2.2).
© Measurement of electroactive species In solution.

@ In order to improve the. sensitivity of electrochemical
assays based on an electrochemical system capable of
generating a current, where the system comprises as compo-
nents an enzyme, a substrate, and a mediator, with the mediator
mediating transfer of electrons between the enzyme and an
electrode while the enzyme is catalysing a reaction of the
substrate, the mediator is indirectly linked with the electrode by
a compound capable of accumulating electrical charge for
measurement after a delay period.

£L
LU

Bundasdnickerei Berlin

0 241 309

Description

MEASUREMENT OF ELECTROACT1VE SPECIES IN SOLUTION

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Background of the Invention

o»I^ S iL nVent !° n re,a J? S t ° J aSSay technic * ues ' more especially to the measurement in solution, for instance

t^Znn°T e °V' qUK l 0r ln " m ° fSt envlronment * surface, of an electroactive species capabte of
transferring charge to or from an electrode. ui

There is a growing interest in the development of novel assay techniques which avoid the use of
radio ■sotope labels In particular, electrochemical methods have been recognised as providing a ve^tfle

m e ~» y f T f< ? r rf " Se ? T ' nCreaSin9 nUmber <* formats - Such assays IncludTa^peremetric
measurement of solution electroactive species. «"Fwuineinc

Snt inve , ntion is Particularly concerned with the measurement of very low concentrations of solution
™S,f P f ' eS ' W *l™ th8 flUX ° f e,eCtrons ,0 or from ™ electrode surface Is t™ XnoS
ToT n^^l 65 10 9ene ^ te 3 meaSUrab ' e CUrrent ™* and above any background electrochemlsW
More particularly, the present invention relates to methods and equipment, involving measurement o

specific subsfrates wrthin the mixture, specific enzymes, or components having a specific binding reaction
acids? q ^

The published European Patent Specification EP78636 (European Application No.82.305597) describes an
assay method for a substrate of an enzyme. The assay is based on the direct electrochemSeaiuTment^

reference! 0 "' "° W referred t0 ,he EP78636> whlch is '""orpoiated herein by

whntS example, EP78636 explains the use of such mediator-linked measurements to detect glucose in
whole blood. To achieve th.s end, a suitable system comprising a mediator and a glucose-catalysing en^ne
(for example glucose oxidase or glucose dehydrogenase) are located on the surface of^Sode^e

a ^l e P l COnta ° ted , Wh0 ' e b ' 00d> md * e SySlem reacts s P eciflca "y ««» th^glose Safbio^d

^ e of mpHL^f "'^'f 1611 10 the 9, ^ C0Se concemratI °n- As mentioned In EP78636. a particularty suitable
for instance ametallocene compound which has electronic properties rendering* capab e
f 9 T'^fr 3 faC " e manner between ,he redox enz y" 1e taction and the electrode Important
^^Lt^ » °T ^ mp ° UndS inc,ude ferrocene and various substituted ferrocene derivatives.
nrf= 0 n,l q " % " "".T real,Zed the technol °9y described In EP78636 might aiso be used to detect the
S ^^fh^T 0 "" 1 ,^ en2yme - such ause, aferrocene or other mediator is used with an excess amoum
with EEES^ ■ "7 ° r dry t sys,em t0 whteh 30 ^"nan <*n be added. The assay specimen will rearf
Tta r«t^,T„^ d Sf" e h rate fcurrent only if asuitable substrate-reacting enzyme is present in the specimen.
The system can be further elaborated to deal with a chain of enzyme reactions -
ouhS e p^Q^°L fUrther ele A ctr ° chemical assay procedures and assay equipment are to be found in
EPI2I1I7 ^iT App ! ,ca ! lon 84.303091), EP125867 (84.303085) EP125136 (84.303086) and

reference. (84 • 303087, • the reader ,s now referred to t" 68 * texts which are incorporated herein by

ra ^* e ^ ° f H * e ! e T t is the use 01 a m adiator to transfer charge while the enzyme is catalysing a
™lci ^ T 3 ^ 6, and - th6 deteoti0n * ,he conse ^antial current which flows. Illustrativetyfora^
enzyme as in the glucose assay ,n EP78636. the assays rely fundamentally on the following cyclical sequences-

substrate

so

55

so

substrate g x '

enzyme 0x <

enzyme Re

mediator

mediator

\

\
\

\ electrode

\
\
s

subslrate"^™ n^^T '° X !.T d ". Re " res P ective| y indi «=ate the oxidised and reduced forms of the
substrate, enzyme or mediator, and *e' indicates an electron. It will be appreciated that the species
"substrateox- is more usually referred to as the product. species
The net effect of the sequence is the continuing transfer of electrons to the electrode which is detected as a
flowing current. The magnitude of the flowing current provides a quantitative measure SeSa^s

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respect, the reader is referred to Examples 7, 8 and 12 of EP78636, and the corresponding Figures 3, 6 and 7
which show the linear correlation between flowing current and glucose concentration.

Essentially, when a steady state enzyme reaction is mediated by a suitable amount of mediator, then a
steady current is obtained. An important further development of the basic system is to be found in EP125139
(84.303090). The EP1 25139 describes the perturbation of the steady state system by effective variations in the 5
amount of enzyme, substrate, mediator, or possibly electrode area. Such pertubations are brought about by a
specific binding reaction, such as an antibody-antigen reaction or such as the reaction of a DNA (or like) probe
with its target sequence. In this way, the EP125139 provides an electrochemical assay for a specific binding
reaction.

In a preferred aspect, the EP125139 is concerned with an improvement in an immunoassay of the kind in 10
which binding of an antigen to an antibody Is assayed by detection of a label. The improvement consists in the
steps of:

providing an electrochemical system capable of generating a current, the system comprising as components

an enzyme, a substrate, and a mediator, with the mediator mediating transfer of electrons between the enzyme

and an electrode while the enzyme is catalysing a reaction of the substrate; 75

employing one of the components as the label; and

detecting the specific binding as an alteration in the electrical current.

A typical and useful example of this improved immunoassay involves setting up a system containing the
enzyme and substrate in a measurement system containing the necessary working electrode and any
reference or counter electrode. There is also present in the system a supply of mediator which is conjugated 20
chemically as a label to an antigen which is in turn linked by a specific binding reaction to an antibody. This
further supply of conjugated mediator is unavailable for electrochemical use in mediation for as long as it
remains linked with the large molecular antigen-antibody complex. Such a total system can be used for
detection or assay of the antigen.

If a specimen containing the antigen is added to the system, some of the antigen will displace the 25
mediator-labelled antigen. The displaced supply of mediator is then in a form conjugated only with the relatively
small antigen molecule, and It can exert its mediating effect. The displaced mediator-labelled antigen can then
transfer electrons between the enzyme and the electrode, thus producing a flowing current proportional to the
amount of free antigen in the specimen.

30

Objects of the Present Invention

The present Invention Is concerned with increasing the sensitivity of the various electrochemical assay
systems described above. A specific object is an increase in the sensitivity of the systems involving specific
binding reactions, whereby very low amounts of the electroactive species (the mediator) can be detected.

For example, the concentration range of many common therapeutic drugs and clinically significant analytes 35
is often in the nanomolar or lower region. A particular object of this invention is a new assay method which
enables detection in the low nanomolar concentration range.

Summary of the present invention

In accordance with the present invention, the mediator is indirectly linked with the electrode by a material 40
capable of accumulating electrical charge for measurement after a delay period.

In a general sense, the invention embraces improvements in the inventions of the European Patents cited
above, where instead of direct mediation by the mediator between the redox enzyme and the electrode to give
rise to a flowing current, the charge is accumulated on a compound which acts as a charge accumulator.

In the present system, a second redox species is used at open circuits as a reagent to accumulate charge 45
during an incubation period from a first redox species acting as mediator. After acting upon the redox enzyme,
the primary mediator is re-oxidlsed or re-reduced by the second redox species. The primary mediator thus
cycles between the second redox species and the redox enzyme/substrate system, generating a pool of
reduced or oxidised second redox species. This pool may then be electrochemlcally measured after a fixed
time. The charge resulting from this step is thus the product of many primary mediator cycles; thus allowing 50
small amounts of primary mediator to be measured as a current, potential or charge, and thus allowing assay of
low levels of analyte.

The electrode can be Interrogated after the fixed incubation time. For instance, such interrogation can be
effected potentiometrically or galvanostaticaliy, and in either case the electrode returned to its original
potential with integration of the resulting current-time transient. The charge accumulated by the electrode and 55
measured by the integration is proportional to the concentration of first redox species mediating the transfer
of electrons between the electrode and the enzyme.

Preferred Embodiments of the Present Invention

Any assay protocol can be employed, including for instance displacement or competitive procedures. The 60
assay of this invention can be performed as a heterogenous or a homogenous assay. The assay is readily
applicable to all kinds of analytes, including drugs, hormones, vitamins, food additives, etc.

The incubation time is best selected through routine experimentation, and typically will be from 10 seconds
to 100 minutes, more typically 5 to 50 minutes and most typically 10 to 20 minutes.

The nature of the substrate and redox enzyme are not criticai, and can be chosen for instance from the 65

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2*^* substmte/enzyme pairings given in the European Patent Specifications mentioned
oxWase^hvZoen nernS s ^. s ^ /on ^ P*™9 * Preferably chosen from glucose/glucose

TtZ n' , hydr0 f 9 t t n Peroxide/horse radish peroxidase; or other suitable well known pairings,
m^orl * » ' S 3)80 " ot cmcaS ' "« <»" be chosen for 'stance from theexamples of suitable

haSnSTete ° r9an ' C med,atO^S • indUdin9 ferr0C6n9 c ° m P° u " d s; phenazine^; carboranes!

illustrafiorthV^Z^ dUS re9a - d iS Paid t0 the nature 01 the second red °* species. By way of

h - • * SPeC ' eS iS P referab| y chosen fr o>" poly(vinytferrocene) or pmssian Uue

se^dTm^s^ 65, 35 ,he char9e <"lator. is noTactive as a mediator, ^d^' be

t ?SSJS '^an ^ "on-mediating byvirtue of physical and/or chemicai proert fes.S
to account the reactivity, electncal charge, molecular size and other factors

h/hf,! red °x species can be non-mediating by immobilization, and then'the assay of this invention win
^?™ S ' Tne se Cond redox species is immob(|ized at ^ WO rkir^ lc roT SpTcS

on the use of a poly(vinylferrocene)-modified electrode. — u,any preierrea assay is based

Thus, for a relatively straightforward heterogenous assay method for detecting redox-active soecies at the
nanomolar concentration level, the method uses an electrode with the second edox specfesTthe chl^a

rh^nl^f w 3 2° ent . ,ometnc 'nterrogation. is returned potentiostatically to its original potential The
22? K 80 d ?'^ ,S i he " 6qUal 10 ,hat acciuired ^ the ^dox-modified electrode during CcubaS
P .n a ^K 96 V t B ' S , dlrect,y P r °P° rtional to *e concentration of redox-active median* ■ i™ esoS
In a particularly preferred arrangement, all the components are on the electrode a foufen I ct^

s?i5;* rtb8d in EP127958 - 71,8 dry strip ^ ctrode 2 ssxsstf^^

SO the^fgtSef h0Ut bei " 9 b ° Und bY ^ Pr6Sent inVenfon ™ be re93rded as «ed by

substrate Re . ^ ^enzyme^ /mediator^ ^ ^redox^

/0

75

a?

55

40

45

50

55

60

65

substrate^/ ^enzyme^/ ^mediatoro,/ ^reda^

cvcln'a^eTpZtifh^ *" se .* ond / ed ° x s P ecies - This charge accumulation occurs with continuing
sXtrlte ' Sed 3,1(1 redUC6d f0m,S 35 me enz y n,B oxidation of the

durina^cum^Hnn of 19 ,! 1131 *" 1™°"* '' nVenti0n ' theflrSt redox s P ecies °° ntinues to act as a mediator
bZeen^iZ^nH a ft™ ^ Sp8Cies - Thus « *» mediatw is continuously cycling

for^Prt ,f reduced forms, as electrons are transferred between the redox species. No convex fe

redox spedes. and there is no similarity of the present invention with the assay
EP155135, where a charge-transfer complex is formed.

r 0 L*T OC H for 3 homogenous assay comprises admixing the substrate, first redox reagent, second redox

wS ££TV H P0,Sm ? t T^ 9 e,eCtf0de: inCUb3tin 3 the °n open circurt re-^felng the

working electrode; and monitonng the current

pre^reoraS 3 meth ° d f ° r detectin9 " measurin 9 or monitori "9 *e

rSf" 0 ^ 3 ? 1 ^ T^ 3 ^ c °mpound * "9"' d medium, by contact with a
torn aTelectrode of a ♦ f*" ™ d ,hU$ Pr0V,din9 ChargB ,0r h^sfer by the mediator to or

nTmTdiator to ftf e ™ ^ f measure ment system: in which a second redox species.

elTcl^e^d in wh^Z!;,^ ' n,ar S 8 excess over the mediator, is also in contact with the

eS ast e ^ir ° f , e ' eCtnC31 Char9e> transferred *«" the mediator to the second redox
species as the enzyme/substrate reaction proceeds, is made after a time interval sufficient to r>ermrt

Z5?3£" 3CCUmul3ti0 " of sucb charge and thus measurement of ,ow K eSSi aS
Schematically, such an immunoassay can be illustrated by the following scheme:

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Ag

substrate

substrate

enzyme w

U Ab

£ Ab:Ag

Ag- mediator^

\ '

enzyme 0x\ /Ag-mediaforpgv ,2nd redox,

Ab:Ag- mediator^

Ag-medbtor^

Ox

2nd redoxRg

w

15

20

In this scheme, the antigen is shown "Ag", the antibody is "Ab" and the antigen-mediator conjugate is
"Ag-mediatorRe" or "Ag-mediatorox". Use of a redox-labelled antigen (which acts as a mediator) in a
competitive immunoassay system allows detection of unlabelled antigen. 25

A redox-modified electrode which is initially, say, fully oxidised can not be reduced at a finite rate by the
solution amplification system, such as glucose oxidase/giucose, except in the presence of a solution small
molecule redox mediator, "Ag-mediator\ The rate at which the electrode modification will be reduced, or the
extent to which it has been reduced after a fixed incubation time, will be in direct proportion to the solution
concentration of active redox mediator. This mediator is the redox-labelled drug conjugate, of which there is a 30
fixed quantity present. It is. however, in competitive equilibrium with unlabelled analyte, Ag, for a fixed number
of solution antibody binding sites, Ab. When a redox-labelled conjugate is bound to antibody, forming
"Ab:Ag-mediator\ it is no longer able to mediate electron flow from glucose oxidase to the redox-modified
electrode surface. Therefore the more unlabelled analyte there is present in the solution the more
redox-labelled analyte there will be available for mediation and hence the more charge there will be 35
accumulated by the redox-modified electron. This charge accumulated during a given incubation time can be
then measured electrochemicaliy by returning the electrode to its original start potential and integrating the
current passed in so doing. The method relies on two reactions not taking place: (i) oxidation of reduced
glucose oxidase by antibody-bound redox-labelled conjugate and (ii) oxidation of glucose oxidase directly by
the redox-modified electrode in the absence of small molecule mediators. In practice, there is no difficulty in 40
ensuring that these reactions do not proceed with appreciable rates.

The invention further provides an electrode system upon which is present one or more of the enzyme,
substrate, mediator, and secondary redox material, together with an immunological or nucleic acid probe
system. Such an electrode may be made up as a dry strip, typically containing a ferrocene-labelled antigen
bound to its antibody, in addition to the enzyme/substrate system and the second redox species. Such an 45
electrode, as part of a suitable measuring system known and described in the earlier European Patent
Applications, can be used to detect the same antigen, which competes for the antibody and in so doing
liberates the ferrocene-labelled antigen, which is then electrochemicaliy available, in low concentrations. If
desired, the electrode systems can be made up so as to contain only some of the species and so as to permit
wet assay using different protocols. 50

The invention will be further described with reference to the following Examples and accompanying
drawings.

Summary of the Drawings

Figure 1 is a calibration curve obtained in Example 2{ii) . 55
Figure 2 is a calibration curve obtained in Example 2(iii).
Figure 3 is a calibration curve obtained in Example 2(iv).

Examples of the Invention

The electrochemical experiments were carried out at room temperature in a two compartment glass cell 60
having a working volume of 1 cm 3 . The working compartment accommodated a 1 cm 2 platinum gauze counter
electrode in addition to the 5mm diameter glassy carbon working electrode. A saturated calomel reference
electrode (SCE) was used in a side arm which connected to the working compartment via a Luggin capillary. A
potentiostat, multimeter, and chart recorder were used to control the potential of the working electrode.
Poised potentials were measured using a high input impedance ( >2 x 10 14 O) programmable electrometer. All 65

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assays were performed under oxygen-free argon. The charge accumulated during an assay was obtained as
the digital integral of the current-time transient resulting when the electrode was scanned linearly with time (10
mV s- 1 ) back to the start potential of + 60GmV: all potentials are with respect to a standard calomel electrode,
"SCE". The transient data were acquired using a digital multimeter interfaced to a microcomputer.

5

Example 1

700 nl of TRIS buffer (0.1M, pH 7.14) containing femcyanide (Fe«i(CN) 1 mMol dm- 3 ) and glucose
(50mMol dm- 3 ) was placed in the sample side of the standard three-electrode electrochemical cell. To this
system the required amount of ferrocene derivative was added (E1/2 < +200 mV). Catalase (2500 U) and
10 glucose oxidase (2000 U glucose oxidase, "GOD", E.C. 1.1.3.4 from Aspergillus niger , RMM 186,000) were
then added. The solution was left in contact with a pyrolytic graphite working electrode on open circuit for 1
minute. The electrode was then poised at +400mV and the i/t transient was recorded. The current at 10
seconds was taken and a dose-response plot constructed. A linear response was obtained.

15 Example 2

Electrocnemically Precipitated Poly(vinylferrocene): an Assay for Theophylline

Pofyfvinylferrocene), "PVF", was prepared essentially according to the method of Smith et ah (Polym Sci
(1976), 14. 2433). The polymer was electrochemicaify precipitated onto 0.2 cm 2 glassy carbon disc electrodes

20 from non-aqueous solutions of 10- 5 M PVF in dichloromethane containing 0.1 M tetra-n-butyl ammonium
perchlorate (J Am Chem Soc (1978), 100, 3222). The deposition potential was typically +700mV and the
immersion time 2 minutes. Upon removal of an electrode from the dichloromethane solution it was shaken to
remove excess solution, sonicated in buffer (20mM potassium phosphate, pH 6.93 plus 0.1 M sodium
perchlorate) is order to remove any loosely held material, and then cycled linearly with time at a rate of 20mV

25 s- 1 between -150 and +650tnV in buffer until a steady voltammogram was obtained. All modified electrodes
were then stored for a minimum of 12 hour in buffer at room temperature before being used.
The mediator was a ferrocene-ethanolamine of formula:

30

35

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The redox-labelled conjugate was 8-ferrocenyl theophylline of formula:

The antibody was anti-theophylline serum Oravenol anti-theo H113), and theophylline was the antigen.
Standard ferrocene-theophylline conjugate solutions were prepared by dilution of a 1mM ethanol stock
solution with buffer.

60

(i) For blank runs containing no mediator conjugate or antibody:

To 500 u.l of 20mM potassium phosphate/0.1 M sodium perchlorate buffer, pH 6.9, was added 50 uJ of
glucose solution (1.30M) followed by 50 uJ of GOD solution (1 .881 mM). After thorough mixing, a PVF modified
glassy carbon electrode was added to the cell, and potentiostatted at + 600mV. At t - 0 minute, the cell was
65 disconnected. After 10 minutes at open circuit the poised potential was measured, the electrode

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re-potentiostatted at this potential and then scanned back to its original potential ( + 600mV), to give a
current-time transient. From this transient the charge accumulated during the 10 minute incubation period was
calculated. These blank runs gave the background response and acted as a control.

(ii) Runs containing varying amounts of mediator conjugate but no antibody: 5
To (550 - x) u.l of buffer was added x u.l of conjugate solution (4.228 x 10- 7 M), followed by 50 uJ of glucose

solution (1.30M) and 50 uJ of GOD solution (1.881mM). After thorough mixing, a PVF-modified electrode was
added to the cell and the assay continued as in (i).

The corrected charge values are plotted against concentration of redox-labelled theophylline conjugate in
Figure 1. w

(iii) Runs containing a fixed amount of conjugate (65nM) and varying amounts of antibody:

To (450 - yj uj'of buffer as added 100uJ of conjugate solution (4.228 x 10- 7 M) followed by y (in the range 15
to 120) uJ of anti-theophylfine serum (diluted 100-fold in buffer), and the solution incubated for 10 minutes at
room temperature. After 10 minutes, 50 uJ of glucose solution (1.30M) was added followed by 50 ul of glucose 15
axidase solution (1.811mM), and the assay continued as in (i).

From the charge-accumulated results of experiments containing 65nM ferrocene-theophylline conjugate
and then Increasing volumes of anti-theophylline serum, it was found that the addition of antiserum resulted in
a decrease in the amount of charge accumulated. The decrease is plotted In Figure 2 as a percentage of the
charge accumulated in a standard experiment containing no added antiserum. The result is consistent with 20
there being a reduction in the amount of redox-labelled conjugate available in solution for mediation as a result
of some having become bound to the added antibodies. A 91 0/0 reduction in the charge normally accumulated
in the presence of 65nM ferrocene-theophylline, glucose and glucose oxidase was observed upon addition of
120uJ of antiserum. Addition of the undiluted anteserum (as supplied) resulted in complete Inhibition of the
catalytic current leading to film reduction with no measurable charge accumulation over and above the 25
background response. Addition of antiserum, theophylline or a mixture of both to standard blank solutions
containing just glucose and glucose oxidase has no measurable effect on the charge accumulated. The results
obtained in each case coincided with the extrapolated background response between successive standard
blank runs.

The results in Figure 2 were used to establish the assay conditions required for the detection of non-labelled 30
theophylline.

(iv) Runs containing fixed amounts of both conjugate and antibody, but varying amounts of theophylline.
To (350-z) uJ of buffer was added 100 uJ of conjugate solution (4.228 x 10- 7 M) followed by z (in the range

100 to 350) uJ of theophylline solution (1.237 u,M). After thorough mixing, 100 u.l of antitheophyliine serum was 35
added and the assay continued as in (iii).

Addition of non-labelled theophylline to runs containing 65nM ferrocene-labelled and a fixed amount of
theophylline antiserum (100 uJ) resulted in a propotional increase in the amount of charge accumulated
compared to that accumulated in the presence of the conjugate and antiserum aione. This increase is due to
the competitive equilibrium set up between the labelled and non-labelled theophylline for a fixed number of 40
antibody binding sites. As the concentration of non-labelled theophylline is increased, so the proportion of
ferrocene-labelled theophylline molecules bound to antibody is decreased. In other words, there is a
proportional increase in the amount of redox-labelled conjugate in soltion available for mediation which results
in a proportional increase in the amount of charge accumulated. It is this relationship between the charge
accumulated and the concentration of added theophylline that forms the basis of the assay. Up to 720/o 45
reversal of Inhibition was achieved upon addition of 666nM theophylline to experimental runs containing fixed
amounts of conjugate (65nM) and antiserum (100 uJ). This corresponded to a 10-fold excess of theophylline
over the labelled theophylline which therefore has somewhat greater affinity for antibody binding sites
compared with theophylline itself. By plotting the amount of charge accumulated during a series of
experimental runs containing varying amounts of added theophylline it was possible to obtain the calibration 50
curve as illustrated in Figure 3. These results show that the system in its present form is capable of
reproducibly detecting theophylline at final concentrations within the range 50-700 nM, with an overall assay
tine of 20 minutes.

Example 3 ^

Electrodeposited Prussian blue

A solution of ferrous sulphate (Fe 2 (S0 4 )3. 20mMol dm- 3 ) and ferricyanide (FeM(CN) 3 -, 20 mMol dm- 3 ) in
10 mM hydrochloric acid was placed in the sample cell of a three electrode electrochemical cell. A polished,
sonicated gold working electrode was contacted with the solution and poised to + 500 mV for 30 seconds. The 60
electrode was then washed and cycled between 0 and +400 mV in 1M potassium chloride for 10 cycles. The
modified electrode was removed and washed, giving a electrode ready for use In the assay. The modified
electrode was placed in a phosphate buffer (20 mM, pH 6.9, 0.1 M KCI) containing glucose (10 mMol dm- 3 )
along with the primary redox active species, a ferrocene (E1/2 < +200 mV). The electrode was poised as
+400 mV and 100 U glucose oxidase added. The electrode was then disconnected and left to accumulate 65

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charge for 10 minutes. After this time the potential of the electrode was measured on the high impedance
voitmeter. The electrode was then poised to this measured potential and swept linearly with time back to + 400
mV. The current-voltage transient was measured and the charge removed from the electrode coat was
evaluated by integration of the area of the transient A linear dose-response curve was again obtained.

5

Example 4

Printed Prussian Blue/Carbon on a Strip
Prussian blue was mixed with a carbon ink at 50 mg/g paste. This was then printed as the working electrode

10 on a two-electrode strip as described in EP127958. Glucose oxidase (100 U) was placed on the surface of the
working electrode, and the sample ferrocene in TRIS buffer (pH 7.2, 100 mMol dm- 3 glucose 0.15 M NaCI) was
added. After 5 minutes the electrode was poised to +350 mV vs Ag/AgCI, and the current-time transient was
recorded. The dose-response curve was linear.
The assay system as described herein is not only generally applicable for use with redox-labeiled conjugates

15 which shuttle charge from the enzyme to the second redox species but it can also be used "in reverse". For
instance, a fully-reduced PVF-modrfied electrode could be used to reduce an enzyme system such as horse
radish peroxidase plus hydrogen peroxide via the intermediacy of the redox-labelled conjugate.

20

Claims

1. An electrochemical assay based on an electrochemical system capable of generating a current,
potential or charge, the system comprising as components an enzyme, a substrate, and a mediator, with

25 the mediator mediating transfer of electrons between the enzyme and an electrode while the enzyme is

catalysing a reaction of the substrate, wherein the mediator is indirectly linked with the electrode by a
compound capable of accumulating electrical charge for measurement after a delay period.

2. The assay of claim 1, wherein said electrical charge is accumulated during an incubation period, and
said electrode is then interrogated.

30 3. The assay of claim 2, wherein for said interrogation, said electrode is returned potentiostatically or

cjafvanostatically to its original potential with integration in either case of the resulting current-time
transient.

4. The assay of claim 1, which is a heterogenous assay and said charge accumulator compound is
immobilized at said electrode.
35 5. The assay of claim 1 , which is a homogenous assay comprising admixing said substrate, a first redox

reagent as said mediator, a second redox reagent as said charge accumulator compound, and said
enzyme; poising said electrode; incubating the system on open circuit; re-poising said electrode; and
monitoring the current.
6. The assay of claim 1 which is an immunoassay.
40 7. The assay of claim 6, where said mediator is a label on an antigen.

8. A dry strip electrode for use in the assay of claim 7 and comprising the enzyme, substrate,
mediator-labelled antigen, charge accumulator compound, and antibody.

9. In an assay of the kind which involves providing an electrochemical system capable of generating a
current, the system comprising as components an enzyme, a substrate, and a mediator, with the mediator

45 mediating transfer of electrons between the enzyme and an electrode while the enzyme is catalysing a

reaction of the substrate; the improvement which consists of the steps of providing a charge accumulator
compound to accumulate a deficit or surfeit of said electrons during an incubation period, and
subsequently monitoring the current represented by said deficit or surfeit

50

55

60

65

8

0241309

CORRECTED

CHARGE ACCUMULATED /^C

6-
4-
2-

i i 1 1 r

15 30 45 60 75

CONCENTRATION Fc THEOPHYLLINE/nN

FIG.1.

% decrease in charge accumulated

20-

40-

60-

80-

100-

i

i i I i —

20 40 60 80
vol. AB added /^l

FIG.2.

100

120

T
90

0241303